Occurrence of arbuscular mycorrhizal fungi in a phosphorus-poor wetland and mycorrhizal response to phosphorus fertilization

The presence of arbuscular mycorrhizas in fens has received little attention, but because fen plants are often phosphorus limited, the plant-fungus interaction could be an important factor in plant competition for phosphorus. In this field study, we determined mycorrhizal colonization rates for 18 fen plant species. Also in the field, we examined the effect of four different forms of phosphorus on the percentage colonization for one fen plant species, Solidago patula. We found that in a species-rich, phosphorus-poor wetland both mycorrhizal and nonmycorrhizal species were common. Nine of ten dicotyledonous species examined formed arbuscular mycorrhizas, while all monocotyledonous species were at most very weakly mycorrhizal. A morphological explanation for this pattern is that the monocots in our study have more extensive aerenchyma, especially in coarse roots. Therefore, monocots are able to transport oxygen to their roots more effectively than dicots. In the organic wetland soil, additional oxygen in the rhizosphere promotes phosphorus mineralization and availability. Two of the monocot species (Typha latifolia and Carex lasiocarpa), which have been described previously as mycorrhizal in other wetland types, are surprisingly nonmycorrhizal in our phosphorus-poor study site, suggesting that a mycorrhizal association would not offer improved phosphorus nutrition to these species. In contrast, our field phosphorus addition decreased mycorrhizal colonization in S. patula, suggesting that one benefit to S. patula of the mycorrhizas is phosphorus uptake.     

Comparison of two test systems for measuring plant phosphorus uptake via arbuscular mycorrhizal fungi

 Plant phosphorus uptake via external hyphae of arbuscular mycorrhizal fungi has been measured using compartmented systems where a hyphal compartment is separated from a rooting compartment by a fine mesh. By labelling the soil within the hyphal compartment with a radioactive phosphorus (P) isotope, hyphal uptake of P into the plant can be traced. The objective of this growth chamber study was to test two hyphal compartments of different design with respect to their suitabilities for measurement of hyphal P uptake. One hyphal compartment was simply a nylon mesh bag filled with ³²P-labelled soil. The labelled soil in the other hyphal compartment was completely surrounded by an 8–10 mm layer of unlabelled soil that served as a buffer zone. Mycorrhizal and non-mycorrhizal subterranean clover plants were grown in pots with a centrally positioned hyphal compartment. Uptake of radioactive P by non-mycorrhizal control plants was 25% of that by mycorrhizal plants with the mesh bag but only 3% when including the buffer zone. Based on this good control of non-mycorrhizal P uptake from within the hyphal compartment and its greater ease of handling once produced, we judged the hyphal compartment including a buffer zone to be superior to the mesh bag. Accepted: 15 September 1998     

Response of strawberry to inoculation with arbuscular mycorrhizal fungi under very high soil phosphorus conditions

A field study was done to assess the potential benefit of arbuscular mycorrhizal (AM) inoculation of elite strawberry plants on plant multiplication, under typical strawberry nursery conditions and, in particular, high soil P fertility (Mehlich-3 extractible P=498 mg kg⁻¹). Commercially in vitro propagated elite plants of five cultivars (‘Chambly,’ ‘Glooscap,’ ‘Joliette,’ ‘Kent,’ and ‘Sweet Charlie’) were transplanted in noninoculated growth substrate or in substrate inoculated with Glomus intraradices or with a mixture of species (G. intraradices, Glomus mosseae, and Glomus etunicatum) at the acclimation stage and were grown for 6 weeks before transplantation in the field. We found that AM fungi can impact on plant productivity in a soil classified as excessively rich in P. Inoculated mother plants produced about 25% fewer daughter plants than the control in Chambly (P=0.03), and Glooscap produced about 50% more (P=0.008) daughter plants when inoculated with G. intraradices, while the productivity of other cultivars was not significantly decreased. Daughter plant shoot mass was not affected by treatments, but their roots had lower, higher, or similar mass, depending on the cultivar–inoculum combination. Root mass was unrelated to plant number. The average level of AM colonization of daughter plants produced by noninoculated mother plants did not exceed 2%, whereas plants produced from inoculated mothers had over 10% of their root length colonized 7 weeks after transplantation of mother plants and ∼6% after 14 weeks (harvest), suggesting that the AM fungi brought into the field by inoculated mother plants had established and spread up to the daughter plants. The host or nonhost nature of the crop species preceding strawberry plant production (barley or buckwheat) had no effect on soil mycorrhizal potential, on mother plant productivity, or on daughter plant mycorrhizal development. Thus, in soil excessively rich in P, inoculation may be the only option for management of the symbiosis.     

Cryopreservation of arbuscular mycorrhizal fungi from root organ and plant cultures

Long-term maintenance of arbuscular mycorrhizal fungi (AMF) by in vitro or in vivo subcultivation is often expensive and time-consuming and could present the risk of contaminations and possibly morphological, physiological, and genetic variations over time. Recently, in vitro produced AMF isolates belonging to the genus Rhizophagus were successfully cryopreserved at ?130 °C following encapsulation-drying. Here, this method was tested on 12 single species cultures belonging to six different genera (i.e., Rhizophagus, Glomus, Claroideoglomus, Septoglomus, Paraglomus, and Gigaspora) produced in vitro or in vivo. Their viability was estimated, after 1 month of cryopreservation at ?130 °C, by the percentage of potentially infective beads (i.e., the percentage of beads that contained at least one germinated propagule) for the in vitro produced species and the percentage of infective beads (i.e., the percentage of beads that contained at least one propagule able to colonize a new host plant in pot culture) for the in vivo produced species. With the exception of Gigaspora sp. MUCL 52331 and Septoglomus constrictus PER 7.2, no significant differences were observed in the viability of the single species cultures before and after cryopreservation. These results, thus, demonstrated the suitability of the cryopreservation method by encapsulation-drying for AMF species belonging to different genera and produced in vitro or in vivo. This method opens the door to the long-term preservation at ultra-low temperature of a large number of AMF species and for the preservation of species that are still recalcitrant to in vitro cultivation.     

Variable responses of old-field perennials to arbuscular mycorrhizal fungi and phosphorus source

If arbuscular mycorrhizal fungi (AMF) promote phosphorus partitioning of plant hosts, they could provide one mechanism for the maintenance of plant community diversity. We investigated whether AMF improved the ability of old field perennials to grow on a range of phosphorus sources and whether AMF facilitated differential performance of plant species on different phosphorus sources (phosphorus niche partitioning). We manipulated form of phosphorus (control versus different inorganic and organic sources) and AM fungal species (control versus four individual AMF species or an AMF community) for five old field perennials grown in a greenhouse in individual culture. Based on biomass after four months of growth, we found no evidence for phosphorus niche partitioning. Rather, we found that effects of AMF varied from parasitic to mutualistic depending on plant species, AMF species, and phosphorus source (significant Plant × Fungus × Phosphorus interaction). Our results suggest that the degree of AMF benefit to a plant host depends not only on AMF species, plant species, and soil phosphorus availability (as has also been found in other work), but can also depend on the form of soil phosphorus. Thus, the position of any AMF species along the mutualism to parasitism continuum may be a complex function of local conditions, and this has implications for understanding plant competitive balance in the field.     

Fenpropimorph and fenhexamid impact phosphorus translocation by arbuscular mycorrhizal fungi

Fenpropimorph and fenhexamid are sterol biosynthesis inhibitor (SBI) molecules widely used to control diseases in agriculture. Both molecules, at increasing concentrations, have been shown to impact on the non-target arbuscular mycorrhizal (AM) fungi. Root colonization, spore production and mycelium architecture, including the branched absorbing structures which are thought to be involved in phosphorus (P) uptake, were affected. In the present study, we investigated the capacity of Glomus sp. MUCL 43204 to take up, transfer and translocate labelled P to Medicago truncatula in the presence of these SBI molecules. We used a strict in vitro cultivation system associating an autotrophic plant of M. truncatula with the AM fungus. In addition, the effects of both SBI molecules on the proportion of hyphae with alkaline phosphatases (ALP), succinate dehydrogenase (SDH) activity and on the expression of the mycorrhiza-specific plant phosphate transporter MtPT4 gene were examined. We demonstrated that the two SBI molecules impacted the AM fungus. This was particularly evidenced for fenpropimorph. A decrease in P transport and ALP and SDH activities associated with the extraradical mycelium and MtPT4 expression level was noted. These three factors were closely related to the development of the AM fungus, suggesting a direct impact not only on the AM fungal growth but also on the physiology and metabolic activities of the AM fungus. These results further emphasized the interest on the autotrophic in vitro culture system as an alternative to pot experiments to investigate the mechanisms behind the impact of disease control molecules on the non-target AM fungal symbionts.     

Trichoderma harzianum might impact phosphorus transport by arbuscular mycorrhizal fungi

Trichoderma sp. is a biocontrol agent active against plant pathogens via mechanisms such as mycoparasitism. Recently, it was demonstrated that Trichoderma harzianum was able to parasitize the mycelium of an arbuscular mycorrhizal (AM) fungus, thus affecting its viability. Here, we question whether this mycoparasitism may reduce the capacity of Glomus sp. to transport phosphorus ((33)P) to its host plant in an in vitro culture system. (33)P was measured in the plant and in the fungal mycelium in the presence/absence of T. harzianum. The viability and metabolic activity of the extraradical mycelium was measured via succinate dehydrogenase and alkaline phosphatase staining. Our study demonstrated an increased uptake of (33)P by the AM fungus in the presence of T. harzianum, possibly related to a stress reaction caused by mycoparasitism. In addition, the disruption of AM extraradical hyphae in the presence of T. harzianum affected the (33)P translocation within the AM fungal mycelium and consequently the transfer of (33)P to the host plant. The effects of T. harzianum on Glomus sp. may thus impact the growth and function of AM fungi and also indirectly plant performance by influencing the source-sink relationship between the two partners of the symbiosis.     

Modelling the contribution of arbuscular mycorrhizal fungi to plant phosphate uptake

In this paper, we investigate the role of arbuscular mycorrhizal fungi in plant phosphorus nutrition. We develop a mathematical model which quantitatively assesses the contribution of external fungal hyphae to plant phosphate uptake.We derive an equation for solute uptake by a growing fungal mycelium which we couple with a model for root uptake. We analyse the model using nondimensionalization and numerical simulations.Simulations predict that removal of phosphate from soil is dominated by hyphal uptake as opposed to root uptake. Model analysis shows that the depletion zones around hyphae overlap within 8 h and that the transfer between fungus and root is a critical step for the behaviour of phosphorus within the mycelial phase. We also show that the volume fraction of mycelium is negligibly small in comparison to other soil phases.This is the first model to quantify the contribution of mycorrhizal fungi to plant phosphate uptake. A full data set for model parametrization and validation is not currently available. Therefore, more complete sets of experimental measurements are necessary to make this model more applicable.     

Distribution of arbuscular mycorrhizal fungi in four semi-mangrove plant communities

To better understand the diversity and species composition of arbuscular mycorrhizal fungi (AMF) in mangrove ecosystems, the AMF colonization and distribution in four semi-mangrove plant communities were investigated. Typical AMF hyphal, vesicle and arbuscular structures were commonly observed in all the root samples, indicating that AMF are important components on the landward fringe of mangrove habitats. AMF spores were extracted from the rhizospheric soils, and an SSU rDNA fragment from each spore morph-type was amplified and sequenced for species identification. AMF species composition and diversity in the roots of each semi-mangrove species were also analyzed based on an SSU-ITS-LSU fragment, which was amplified, cloned and sequenced from root samples. In total, 11 unique AMF sequences were obtained from spores and 172 from roots. Phylogenetic analyses indicated that the sequences from the soil and roots were grouped into 5 and 14 phylotypes, respectively. AMF from six genera including Acaulospora, Claroideoglomus, Diversispora, Funneliformis, Paraglomus, and Rhizophagus were identified, with a further six phylotypes from the Glomeraceae family that could not be identified to the genus level. The AMF genus composition in the investigated semi-mangrove communities was very similar to that in the intertidal zone of this mangrove ecosystem and other investigated mangrove ecosystems, implying possible fungal adaptation to mangrove conditions.     

Myco-heterotroph/epiparasitic plant interactions with ectomycorrhizal and arbuscular mycorrhizal fungi

More than 400 achlorophyllous plant species in 87 genera are parasitic upon fungi, and exploit them as their principle source of carbon. With a few exceptions, most of these myco-heterotrophic plants are now thought to be 'cheats', stealing carbon and nutrients from the mycorrhizal associates of adjacent autotrophic plants. Most myco-heterotrophs are therefore considered to be epiparasitic on green plants. Both the ectomycorrhizal and arbuscular mycorrhizal symbioses have been invaded by myco-heterotrophic epiparasites. DNA analysis is revealing the identities of many of the fungal partners of myco-heterotrophs, and their exceptionally high specificity. Myco-heterotrophs have distinctive stable isotope signatures, which can be used to establish the dependence upon fungal carbon of green plants that are partially myco-heterotrophic.     

Towards growth of arbuscular mycorrhizal fungi independent of a plant host

When surface-sterilized spores of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices Sy167 were germinated on agar plates in the slightly modified minimum mineral medium described by G. Bécard and J. A. Fortin (New Phytol. 108:211-218, 1988), slime-forming bacteria, identified as Paenibacillus validus, frequently grew up. These bacteria were able to support growth of the fungus on the agar plates. In the presence of P. validus, hyphae branched profusely and formed coiled structures. These were much more densely packed than the so-called arbuscule-like structures which are formed by AMF grown in coculture with carrot roots transformed with T-DNA from Agrobacterium rhizogenes. The presence of P. validus alone also enabled G. intraradices to form new spores, mainly at the densely packed hyphal coils. The new spores were not as abundant as and were smaller than those formed by AMF in the monoxenic culture with carrot root tissues, but they also contained lipid droplets and a large number of nuclei. In these experiments P. validus could not be replaced by bacteria such as Escherichia coli K-12 or Azospirillum brasilense Sp7. Although no conditions under which the daughter spores regerminate and colonize plants have been found yet, and no factor(s) from P. validus which stimulates fungal growth has been identified, the present findings might be a significant step forward toward growth of AMF independent of any plant host.     

Genetic processes in arbuscular mycorrhizal fungi

Arbuscular mycorrhizal (AM) fungi (Glomeromycota) colonize roots of the majority of land plants and facilitate their mineral nutrient uptake. Consequently, AM fungi play an important role in terrestrial ecosystems and are becoming a component of sustainable land management practices. The absence of sexual reproductive structures in modern Glomeromycota combined with their long evolutionary history suggest that these fungi may represent an ancient asexual lineage of great potential interest to evolutionary biology. However, many aspects of basic AM fungal biology, including genome structure, within-individual genetic variation, and reproductive mode are poorly understood. These knowledge gaps hinder research on the mechanisms of AM fungal interactions with individual plants and plant communities, and utilization of AM fungi in agricultural practices. I present here the current state of research on the reproduction in AM fungi and indicate what new findings can be expected in the future.     

Response of endangered plant species to inoculation with arbuscular mycorrhizal fungi and soil bacteria

Three endangered plant species, Plantago atrata and Pulsatilla slavica, which are on the IUCN red list of plants, and Senecio umbrosus, which is extinct in the wild in Poland, were inoculated with soil microorganisms to evaluate their responsiveness to inoculation and to select the most effective microbial consortium for application in conservation projects. Individuals of these taxa were cultivated with (1) native arbuscular mycorrhizal fungi (AMF) isolated from natural habitats of the investigated species, (2) a mixture of AMF strains available in the laboratory, and (3) a combination of AMF lab strains with rhizobacteria. The plants were found to be dependent on AMF for their growth; the mycorrhizal dependency for P. atrata was 91%, S. umbrosus-95%, and P. slavica-65%. The applied inocula did not significantly differ in the stimulation of the growth of P. atrata and S. umbrosus, while in P. slavica, native AMF proved to be the less efficient. We therefore conclude that AMF application can improve the ex situ propagation of these three threatened taxa and may contribute to the success of S. umbrosus reintroduction. A multilevel analysis of chlorophyll a fluorescence transients by the JIP test permitted an in vivo evaluation of plant vitality in terms of biophysical parameters quantifying photosynthetic energy conservation, which was found to be in good agreement with the results concerning physiological parameters. Therefore, the JIP test can be used to evaluate the influence of AMF on endangered plants, with the additional advantage of being applicable in monitoring in a noninvasive way the acclimatization of reintroduced species in nature.     

Increasing phosphorus removal in willow and poplar vegetation filters using arbuscular mycorrhizal fungi

Fast growing woody species are increasingly used in vegetation filters for wastewater treatment. Their efficiency in phosphorus (P) removal notably depends on plant uptake and storage in aboveground tissues. In this study, Populus NM5 (P. nigra × P. maximowiczii), Salix miyabeana (SX64) and Salix viminalis (5027) were planted in pots to evaluate the influence of colonization by arbuscular mycorrhizal fungi (AMF) Glomus intraradices on P uptake using two different P concentrations in irrigation water. Based on analysis of the foliar and woody components, our results show that the two treatments (inoculation with G. intaradices and P-irrigation) interact differently with total P content. Foliar P content is principally enhanced by the P-irrigation concentration, whereas the mycorrhizal colonization increases stem P content. In the presence of G. intraradices, both S. miyabeana and S. viminalis showed a 33% increase in stem P content. The latter finding is mainly due to an increase in biomass production, without modification of the P concentration, indicating that AMF associations affect P use efficiency. Thus, using arbuscular mycorrhizal fungi for phytoremediation strategies may increase biomass productivity and hence improve pollutant uptake.     

Biolistic transformation of arbuscular mycorrhizal fungi

Gene transfer systems have proved effective for the transformation of a range of organisms for both fundamental and applied studies. Biolistic transformation is a powerful method for the gene transfer into various organisms and tissues that have proved recalcitrant to more conventional means. For fungi, the biolistic approach is particularly effective where protoplasts are difficult to obtain and/or the organisms are difficult to culture. This is particularly applicable to arbuscular mycorrhizal (AM) fungi, being as they are obligate symbionts that can only be propagated in association with intact plants or root explants. Furthermore, these fungi are aseptate and protoplasts cannot be released. Recent advancements in gene transformation systems have enabled the use of biolistic technology to introduce foreign DNA linked to molecular markers into these fungi. In this review we discuss the development of transformation strategies for AM fungi by biolistics and highlight the areas of this technology which require further development for the stable transformation of these elusive organisms.     

Effect of long-term tillage and mineral phosphorus fertilization on arbuscular mycorrhizal fungi in a humid continental zone of Eastern Canada

Aims

Evidence shows that tillage modifies soil properties, especially phosphorus (P) dynamics. Our objective was to disentangle long-term effects of P-fertilization and tillage on arbuscular mycorrhizal fungal (AMF) proliferation and community structure.

Methods

Changes in the community structure of AMF and in the density of their hyphae and spores induced by moldboard plow (MP) or no till (NT), and fertilization with 0, 17.5, or 35 kg?P?ha^?1 were sought in the 0–15 cm and 15–30 cm soil layers after soybean harvest, at a long-term (17 years) experimental site in a humid continental zone of eastern Canada. The relationships among AMF, soil and plant attributes were examined.

Results

The 0–15 cm and 15–30 cm soil layers had different properties under NT, but were similar under MP, after 17 years, and MP increased soil available P levels. Phosphorus fertilization increased P levels in soil and in soybean. Treatment effects on AMF spore and hyphal density at 0–15 cm were greater than that at 15–30 cm, whereas effects on AMF community structure did not change with soil depths. At 0–15 cm, P-fertilization increased AMF spore density and reduced AMF hyphal density, and MP reduced AMF spore density. A total of eight AMF phylotypes were detected. Phosphorus fertilization reduced AMF phylotype richness and Shannon diversity index. Soil P availability increased under MP and hence the influence of P-fertilization treatments on the frequency of AMF phylotype detection varied with tillage system; it declined with P-fertilization under MP, but increased under NT.

Conclusions

Phosphorus fertilization shifts resource partitioning in AMF propagules rather than in their hyphae, and degrades the genetic diversity of AMF in soil; tillage increases soil P availability and hence aggravates the impact of P-fertilization.